Why is the actual band size on a western blot different from the predicted size of the protein?
Western blotting is based on the separation of proteins by their size on a gel. However, migration of proteins through the gel matrix is also affected by other factors, which may cause the observed band size to be different from the predicted size.
Common causes are:
-Post-translational modification; for example phosphorylation and glycosylation increase the size of the protein
-Post-translation cleavage; many proteins are synthesized as precursor proteins, and then cleaved to give the active form
-Multimers, for example dimerization of a protein. This is usually prevented under reducing conditions, although strong interactions can result in the appearance of higher bands
-Splice variants; alternative splicing may result in different sized proteins being produced from the same gene
-Relative charge; the composition of amino acids (charged vs. non-charged)